Methods for the isolation and expansion of cord blood derived T regulatory cells

ABSTRACT

The present invention encompasses methods, and kits for the isolation and expansion of T regulatory cells having the CD45RA +  phenotype, including such cells from human umbilical cord blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority pursuant to 35 U.S.C. §119(e) toU.S. Provisional Application No. 60/609,916, filed Sep. 15, 2004, whichis hereby incorporated by reference in its entirety herein.

GOVERNMENT INTERESTS

This invention was supported in part by the National Institutes ofHealth Grant Nos. R01 A134495 and R37 HL56067. The Government may havecertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to T regulatory (T_(reg)) cells isolatedfrom human cord blood, as well as compositions, methods and kits usingT_(reg) cells so isolated.

BACKGROUND OF THE INVENTION

Naturally arising CD4⁺CD25⁺ T regulatory cells (T_(reg)) can restrict oralter most types of immune responses (Sakaguchi, 2004, Annu. Rev.Immunol., 22: 531-562). Initially, T_(reg) cells were described to becritical for the control of autoimmunity (Sakaguchi, et al., 1995, J.Immunol., 155: 1151-1164; Shevach, 2000, Annu. Rev. Immunol., 18:423-449), and were found on adoptive transfer to prevent experimentalautoimmune diseases. More recently, T_(reg) have been shown to suppressallogeneic immune responses, and can prevent transplant rejection (Hall,et al, 1998, J. Immunol., 161: 5147-5156; Wood, et al., 2003, Nat. Rev.Immunol., 3: 199-210). In addition, these cells can restrain anti-tumor(Peng L, et al., 2002, J. Immunol., 169: 4811-4821; Gallimore, et al.,2002, Immunology, 107: 5-9), and anti-microbial immune responses(Belkaid Y, et al., 2002, Nature, 420: 502-507). Thus, CD4⁺CD25⁺ T_(reg)appear to be central control elements of immunoregulation, andunderstanding their biology is important to efforts aimed attherapeutically manipulating immune responses. T_(reg) cells are bestcharacterized in mice where they constitute 5-10% of lymph node andspleen CD4⁺ T-cell populations. They are generated both through centralthymic developmental mechanisms in pathogen free mice, and also arise bypoorly defined peripheral generation or expansion mechanisms (Apostolou,et al., 2002, Nat. Immunol., 3: 756-763; Shevach et al., 2002, Nat. Rev.Immunol., 2: 389-400). To date, T_(reg) cells have primarily beendefined by co-expression of CD4⁺ and CD25⁺ antigens on fresh isolation.CD25 as well as other markers of murine T_(reg), CTLA4 (CD 152) and GITR(Glucocorticoid Induced TNF-like Receptor), are all activation antigenson conventional T cells, and therefore are not specific. FoxP3, anuclear protein thought to function as a transcriptional repressor, is anewer marker considered to be more specific for T_(reg) cells (Ramsdell,et al., 2003, Curr. Opin. Immunol., 15: 718-24). It was demonstratedthat after activation (T cell receptor based, antigen-specific oranti-CD3), T_(reg) cells can non-specifically suppress proliferation ofboth CD4⁺ and CD8⁺ T cells. The mechanism of suppression is unclear, andin vitro, appears to require cell-cell contact. A functional result ofsuppression is impaired production of IL-2 (Thornton, et al., 1998, J.Exp. Med., 188: 287-296; Shevach, et al., 2001, Immunol. Rev., 182:58-67). In vivo, the suppression mechanism is more controversial withsome studies demonstrating dependence on immunosuppressive cytokines(Asseman, et al., 1999, J. Exp. Med., 190: 995-1004), which are notrequired for in vitro suppression.

Studies in mouse models of bone marrow transplantation (BMT) have shownthat fresh or culture expanded CD4⁺CD25⁺ cells can delay or preventdisease (Taylor et al., 2002, Blood, 99: 3493-3499″ Hoffmann, et al.,2002, J. Exp. Med., 196: 389-399; Cohen, et al., 2002, J. Exp. Med.,196: 401-406). Previous studies have demonstrated that T_(reg)polyclonally expanded ex vivo for 10 days with anti-CD3 plus IL-2, canbe effective in preventing graft versus host disease (GVHD; Taylor, etal., 2002, Blood, 99: 3493-3499). Ex vivo expansion of T_(reg) cellswith irradiated allogeneic APCs plus exogenous IL-2 is also effective atsuppressing GVHD (Cohen, et al., 2002, J. Exp. Med., 196: 401-406). Insome model systems, T_(reg) cells can prevent GVHD and still allow forgraft versus leukemia (GVL) effects (Edinger, et al., 2003, Nat. Med.,9: 1144-1150; Jones, et al., 2003, Biol. Blood Marrow Transplant, 9:243-56; Trenado, et al., 2003, J. Clin. Invest., 112: 1688-96). Inaddition, studies in mouse models of autoimmune disease havedemonstrated that culture expanded antigen specific (transgenic TCR)CD4⁺CD25⁺ cells can prevent or even treat diabetes (Tang, et al., 2004,J. Exp. Med., 199: 1455-1465). Consequently, T_(reg) cells have a rolein clinical immunosuppressive therapy in transplantation, provided humanT_(reg) cells can be isolated and expanded in culture to generatesufficient numbers for in vivo infusion.

While the murine data are very promising, there still remains apractical problem of isolating pure T_(reg) from human blood. In youngmice, CD4⁺CD25⁺ cells are moderately abundant and the CD25⁺ subset isreadily apparent. In humans the CD25⁺ cells are not as discrete of apopulation, as there exists a large and overlapping population ofCD25-dim cells. It is possible that the co-purification of conventionalT cells with T_(reg) is the basis for the modest or variable suppressoractivity observed in studies of human CD4⁺CD25⁺ cells (Baecher-Allan, etal., 2004, Semin. Immunol., 16: 89-98). FACS cell sorting of the highest1.7% of CD25⁺ expressors (CD25^(high) cells) has been reported to enablesuppressor cell isolation (Baecher-Allan, et al., 2001, J. Immunol.,167: 1245-1253). A stringent magnetic bead based approach was requiredto isolate populations of adult blood derived T_(reg) cells pure enoughfor CD4⁺CD25⁺ cells to generate potent suppressor cell lines. Even so,strongly suppressive cell lines could only be generated in a subset(approximately one third) of donors, and potency correlated with cellline purity (Godfrey, et. al., 2004, Blood, 104: 453-461). FACS sortingof CD25^(high) cells (top 2.1%) has been reported to enable moreconsistent suppressor cell line generation from adult blood (Hoffmann,et al., 2004, Blood. 104: 895-903).

Purification of T_(reg) cells from adult blood is possible, butdifficult. Previous attempts using magnetic activated cell sorting(MACS) purification to isolate T_(reg) cells from adult blood that aresufficiently pure for consistent suppressor activity have resulted invariability in cell function. This variability is largely due to thepresence of CD25-dim memory cells which overlap with T_(reg) cells. Useof a cell sorter has facilitated the isolation of T_(reg) cells(Baecher-Allan, et al., 2001, J. Immunol., 167: 1245-1253), and enabledthe generation of suppressor cell lines from adult blood (Hoffmann, etal., 2004, Blood, 104: 895-903). However, even sorted populations ofadult blood derived CD25⁺ cells (top 2.9%) were found in one report tocontain a mix of conventional and regulatory T cells on cloning andfunctional analysis (Levings, et. al., 2002, J. Exp. Med., 196:1335-1346).

About 20% of of the CD4⁺CD25⁺ adult blood cells express CD45RA. Thisantigen is not expected to be expressed on suppressor cells, as theyhave been described in several reports to be CD45RO positive (generallymutually exclusive expression, except for transiently during activationof naive cells). However, the isolation of these cells was much betterthan the CD45RA⁻ cells for generating suppressor cell lines (12/12 celllines isolated by this method were found to be potent suppressors). Onnaive T cells the CD45RA splice variant is expressed on the T cellsurface. Once a T cell differentiates into a memory cell, it usuallyexpresses the CD45RO isoform (U.S. Publication No. 20050196386).

Cord blood has previously been shown to contain CD4⁺CD25⁺ cells byfluorescence activated cells sorting (FACS) (Paganelli, et. al., 1994,Cell Immunol., 155: 486-489; Ng, et al., 2001, Blood 98: 2736-2744;Wing, et al., 2002, Immunology 106: 190-199). However, there is minimaldata reported on the function of these cells. One report has inferredsuppressive function based on LDA frequency analysis (Ng, et al., 2001,Blood 98: 2736-2744). The only report evaluating functional activity offreshly isolated CD4⁺CD25⁺ cells, revealed no suppression of antigenspecific responses. In addition, there was no increased antigen specificreactivity of CD4⁺ cells after CD25⁺ cell depletion. However, modestsuppression was noted in anti-CD3 based T-cell co-culture assays, (60%at 1/1 responder/suppressor cell ratio) (Wing, et al., 2003, Eur. J.Immunol., 33: 579-587). Thus, previous studies indicated that most cordblood derived CD25⁺ cells were not yet mature enough to be suppressive(Wing K, et al., 2003, Eur. J. Immunol. 33: 579-587).

Accordingly, until the present invention, the properties and benefits ofT_(reg) cells were recognized, but method to isolate and generatesufficient numbers of potent suppressor cells were unknown. Therefore, arecognized need for methods to isolate and expand T_(reg) cells existed.The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention includes a method for isolating a regulatory Tcell from a population of phenotypically CD45RA⁺ blood cells, whereinthe T_(reg) cell suppresses T cell proliferation. The method of thepresent invention comprises isolating a population of mononuclear cellsfrom a human umbilical cord blood sample, contacting the population ofmononuclear cells with an antibody that specifically binds CD25 underconditions suitable for formation of a mononuclear cell-antibodycomplex, and substantially separating the mononuclear cell-antibodycomplex from said population of mononuclear cells, thereby isolating aregulatory T cell from a population of phenotypically CD45RA⁺ bloodcells. In one embodiment of the invention, the population ofphenotypically CD45RA⁺ blood cells is from umbilical cord blood,preferably a human umbilical cord sample.

The present invention further includes a method of multiplying anisolated regulatory T cell comprising culturing the regulatory T cell ina medium comprising an antibody to CD3 and an antibody to CD28. Themedium can further comprise IL-2.

The present invention further comprises a method of inhibitingproliferation of a T cell. The method comprises contacting a T cell witha regulatory T cell isolated by the method described herein.

The present invention also includes kit for isolating a regulatory Tcell from a human umbilical cord blood sample. The kit comprises anantibody that specifically binds CD25 bound to a physical support, anapplicator, and an instructional material for the use thereof.

The present invention also includes kit for multiplying a T_(reg) cellfrom a human umbilical cord blood sample. The kit comprises an antibodythat specifically binds CD3 bound to a physical support, an antibodythat specifically binds CD28 bound to a physical support, an applicator,and an instructional material for the use thereof.

The present invention further comprises a regulatory T cell isolated bythe methods of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are depicted in the drawings certain embodiments of the invention.However, the invention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings. All errorbars represent one standard deviation above and below the mean.

FIG. 1, comprising FIGS. 1A through 1D, is a series of images depictingFACS analysis for CD45RA and CD45RO expression and optimum purificationsof CD45RA⁺ and CD45RO⁺ cells.

FIG. 2 is an image depicting that CD45RA⁺ cells do not express CD25 ateven the highest levels, and that CD25 bright cells do not detectablyexpress CD45RA.

FIG. 3, comprising FIGS. 3A and 3B, is a series of images depictingpotent suppressor activity in CD45RA⁺ cells as measured by a mixedlymphocyte reaction.

FIG. 4, comprising FIGS. 4A through 4D, is a series of images depictingthat CD45RA cells are CTLA-4 negative (FIG. 4A) and HLA-DR⁻ (FIG. 4B).CD25⁺ bright cells are HLA-DR⁺ (FIG. 4C) and they double stain forintracellular CTLA-4, thus depicting two distinct cell subsets.

FIG. 5 is a graph depicting the suppressor activity of CD45RA⁺ cells(RA+), HLA-DR⁺ cells (DR+) and double negative cells (DN). The mostpotent suppressor activity is evident in CD45RA⁺ cells, middle potencyin double negative cells and the least potency in HLA-DR⁺ cells.

FIG. 6, comprising FIGS. 6A through 6D, is a series of images depictingthat CD4⁺CD25⁺ cord blood cells are a distinct population. FIG. 6comprises representative FACS plots of peripheral blood mononuclearcells (PBMC), cord blood mononuclear cells (CBMC) and purified CD4⁺CD25⁺cells from both sources. FIG. 6 is representative of 10 donors and 10cell purification experiments. FIG. 6A depicts distinct populations ofCD4⁺ and CD25⁺ cells in cord blood with a wide separation of CD25⁺ cellsfrom CD25⁻ cells. FIG. 6B is an image depicting that CD4⁺CD25⁺ cellsconstitute 1-2% of PBMC, and that a large number of these cells areCD25-dim (arrow). FIG. 6C is an image depicting that CD25⁺ cellspurified from cord blood by direct anti-CD25-microbeads are a purerpopulation. FIG. 6D is an image depicting CD25⁺ cells purified fromadult blood by direct anti-CD25-microbeads.

FIG. 7 is an image depicting that cord blood CD25⁺ cells are CD45RA⁺ andshowing that purified CD25⁺ cord blood cells are predominantly CD45RA⁺.

FIG. 8, comprising FIGS. 8A through 8D, is a series of imagesdemonstrating that cultured cord blood derived CD4⁺CD25⁺ cells markedlysuppress a mixed lymphocyte reaction (MLR) as measured by proliferativeinhibition. FIG. 8A is a graph depicting the kinetic curves ofproliferation over a one week MLR. Cord blood derived cells essentiallyblock MLR (●), adult cell lines selected directly by MACS have weaksuppressor function (□), and stringently selected adult cells(CD25⁺⁺lineage-) have moderate potency (▪). CD25⁻ cells are notsuppressive (*). FIG. 8A is representative of 10 experiments. FIG. 8B isa scatter plot demonstrating the consistency of suppression at day 6 ofMLR of cord blood derived (●), versus direct MACS adult cell lines (□),or stringently purified adult lines (CD25⁺⁺lineage-) (▪). FIG. 8C is agraph depicting graded numbers of cultured T_(reg) cells added to an MLRreaction to determine the minimum number needed for potent inhibition.Up to a 1:32 dilution (roughly 1,560 suppressors/50,000 responders)markedly impaired MLR when using cord blood derived suppressor celllines (●), versus 1:16 for selected potent subset of stringentlypurified adult lines (pCD25⁺⁺lin-) (Δ). Two lines each are depicted,representative of 6 adult and cord blood derived suppressor cell lines.FIG. 8D is a graph depicting the maturation of dendritic cells (DC)prior to MLR, by lipopolysaccharide (LPS) or TNF/polyIC combination.Inclusion of these stimulating factors in MLR fails to bypasssuppression. FIG. 8D is representative of 3 experiments.

FIG. 9, comprising FIGS. 9A through 9B, is a series of imagesdemonstrating that cultured CD4⁺CD25⁺ cells markedly suppress cytokineaccumulation in MLR, as analyzed by assessment of cytokine levels. FIG.9A is a graph depicting an impairment in the accumulation of cytokinesproduced by activated T cells, specifically IL-2, IFN-gamma, GM-CSF,TNF-alpha, IL-5, and IL-10 is observed. No alteration in TGF-beta 1accumulation is detectable. FIG. 9A depicts IL-2 levels on day 2, andother cytokines on day 6, the respective times of peak of accumulationin control MLR cultures. FIG. 9B is a graph depicting minimal alterationof chemokine levels at early timepoints (Day 2), and modest decreases inlevels at late time-points (Day 7) for rantes, IL-8, and MIP-1a. FIG. 9is representative of 4 MLR experiments.

FIG. 10, comprising FIGS. 10A and 10B, is an image depictingrepresentative plots of a flow cytometric comparison of CD25⁺, CD25⁻,and CD25-derived cell lines after 3-4 weeks culture expansion. FIG. 10Ais an image demonstrating that CD25 and intracellular CTLA4 expressionremains high on cord blood T cells, expression is lower in adult derivedT cells, and expression largely returns to baseline for the CD25-derivedcell lines. FIG. 10B is an image depicting that CD62L and CD27expression remains uniformly high on cord blood T cell lines, also on asubset of the adult lines, and diminishes on the CD25-derived celllines. FIG. 10 is representative of 10 cell lines each, analyzed at 4weeks of culture.

FIG. 11, comprising FIGS. 11A and 11B, is an image depicting FoxP3 mRNAand protein expression. FIG. 11 A is a graph depicting levels of FoxP3mRNA assessed by real time PCR analysis. Samples are freshly isolatedcells (fresh CD25), cell lines after 4 weeks of culture (Cx CD25), and 4week cultured cell lines 24 hours after re-stimulation withanti-CD3/CD28 beads (Restim CD25), as indicated. Data are plotted asfold comparison to mRNA levels present in freshly isolated CD8⁺ T cells(CD8). FIG. 11B is a series of images of a western blot analysis ofFoxP3 protein expression in cells cultured for 4 weeks and restimulatedwith anti-CD3/CD28 beads for the time indicated. Histone H3 was used asa loading control for nuclear proteins. FIG. 11 is representative of 4independent experiments.

FIG. 12, comprising FIGS. 12A through 12D, is a series of imagesdepicting cytokine production defects and cell surface LAP expression 48hours after re-stimulation of suppressor lines. FIG. 12A is a graphdepicting cell lines reactivated with anti-CD3/CD28 beads. FIG. 12B is agraph depicting CD25⁻ and CD25⁺ cells reactivated with PMA/ionomycin.FIG. 12C is an image depicting expression of CD69, OX40(CD 134), andGITR intact. FIG. 12D is an image depicting that cell surface LAPexpression is specific for CD25⁺ derived cell lines. Resting cell lineswere negative for these activation antigens prior to re-stimulation.

FIG. 13, comprising FIGS. 13A through 13D, is a series of imagesdepicting a screen of multiple neutralizing antibodies and fusionproteins for functional effects on the suppression of a CD4⁺ T cell-DCMLR. FIG. 13A is a graph depicting that antibodies to immunosuppressivefactors IL-10 and TGF beta, as well as anti-IL10R, or combinations ofall three fail to reverse suppression mediated by cultured T_(reg) celllines. FIG. 13B is a graph depicting that antibodies to negativesignaling molecules, CTLA4 and PD1, do not reverse suppression. FIG. 13Cis an image depicting the lack of suppression from antibodies to GITR orGITR-L. Agonist antibodies to OX40 partially reverse suppression whileantibody to OX40L partially enhances suppression. FIG. 13D is a graphdepicting that multiple TGF-beta neutralizing antibodies and solublereceptors do not prevent suppressor cell effector function. FIGS. 13Athrough C are representative of 6 independent experiments, FIG. 13D isrepresentative of 3 independent experiments.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention encompasses methods and kits for the isolation andexpansion of an enhanced population of T regulatory cells (T_(reg))having the CD45RA⁺ phenotype. The term “enhanced”, as used herein refersto at least 20% more, preferably 30%, preferably 40%, even morepreferably 50%, even more preferably about 60%, even more preferably60%, even more preferably 70%, even more preferably 80%, even morepreferably 90%, and even more preferably 100% more than an object thatis not enhanced. As an example, an enhanced population of regulatorycells has at least 20% more regulatory cells than a population that isnot enhanced. The preferred source of such cells was found to be humanumbilical cord blood. Methods for producing large numbers of active,potent suppressor T_(reg) cells from other sources, such as adult humanblood, have been confounded by the presence of memory T cells expressingCD25 at low to moderate levels (CD25dim cells), and conventionalactivated T-cells (CD25⁺) that interfere with T_(reg) cell purification.The term “T_(reg)” is used herein to refer to a regulatory T cell thatexpresses both CD4 (CD4⁺) and CD25 (CD25⁺). However, as demonstrated bythe data disclosed herein, both of these types of T cells (memory andactivated) are generally lacking, or in lower numbers, in umbilical cordblood. This is because, as disclosed herein, cord blood cells develop ina protected environment and are immunologically naive. Therefore, thepresent invention demonstrates that the CD25⁺ T_(reg) cells in umbilicalcord blood are more readily purified than those derived from othersources, such as human adult peripheral blood. A cell that is “CD25⁺” orthat “expresses CD25” is contrasted herein to a cell that is CD25⁻ ordoes not express a detectable level of CD25. A cell that is “CD25dim,”as used herein has a lower detectable level of CD25 expression than aCD25⁺ cell or a “CD25bright” cell. In addition, culturing andmultiplying these cells is facilitated and simplified because feedercells are not necessary to expand T_(reg) cell populations.

T_(reg) cells are critical to self and allograft tolerance in mice.Studies of human T_(reg) have been hindered by low numbers present inperipheral blood and difficult purification. The data disclosed hereindemonstrate that cord blood is a superior source for T_(reg) isolationand cell line generation compared to adult blood. Cord blood CD4⁺CD25⁺cells were purified, and cell lines were generated that consistentlyexhibited potent suppressor activity, with >95% suppression ofallogeneic MLR (29/30 donors). Cultured T_(reg) cells blocked cytokineaccumulation in MLR, with inhibition of chemokine production. These celllines uniformly expressed CD25, CD62L, CCR7, CD27, and intracellularCTLA4. Further, FoxP3 protein, but not mRNA, was specifically expressed.Upon re-stimulation with anti-CD3/CD28 beads, the cultured T_(reg)produced minimal cytokines (IL-2, IFN-gamma, and IL-10), andpreferentially expressed TGF-beta latency associated protein. Cytokineproduction however, was restored to normal levels by re-stimulation withPMA/ionomycin. Cord blood derived cultured suppressor cell function waspredominantly independent of IL-10 and TGF-beta. The data disclosedherein demonstrate that cord blood contains a significant number ofT_(reg) precursor cells, capable of potent suppressor function afterculture activation. Banked cord blood specimens may serve as a readilyavailable source of T_(reg) for immunotherapy.

The present invention further encompasses T_(reg) cells isolated fromhuman umbilical cord blood samples that have more potent suppressoractivity and cytokine suppression capabilities than those derived fromother sources. In marked contrast to the prior art, the data disclosedherein demonstrates that T_(reg) cells isolated from human umbilicalcord blood have a potent ability to suppress T cell proliferation and tosuppress T cell activation-dependent cytokines. Further, the presentdata further demonstrates that cord blood CD4⁺CD25⁺ cells can formpotent suppressor cells after isolation and culture. The presentinvention encompasses a method for purifying T_(reg) cells using astraightforward direct antibody-based purification system for isolatingsuch suppressor cells. After activation and multiplication withanti-CD3/CD28 beads and culture in IL-2, cord blood derived CD4⁺CD25⁺cells acquire potent suppressor function, which was maintained for longperiods of time. Thus, the present invention further encompasses amethod for inhibiting T cell proliferation and a method for inhibitingcytokine production.

To date, all assays of suppressor cell phenotype and function revealthat the cord blood derived, and the purest of the adult derived cells,have similar profiles. Flow cytometric analysis, cytokine productionpotential, and functional profiling have all been essentially the same.Thus, the data disclosed herein indicate that both types of lines, whenpure, express equivalent suppressor mechanisms and potency, and theadvantage of cord blood primarily relates to ease of purification andculture.

The availability of large numbers of suppressor cells has enabled thebiochemical and molecular characterization of these special cells. Usingthe methods disclosed herein, greater than 300 million uniform andpotent suppressor cells can be generated from one average sized researchgrade cord blood unit (˜300 million cells) without a cell sorter. Theseresults demonstrate a 1% CD4⁺CD25⁺ cell recovery, and a 100-foldexpansion in culture. This number of cells enables furthercharacterization of TCR signaling alterations, FoxP3 regulation andfunction, and suppressor effector mechanisms. In addition, an importantutility of these cells is to enable clinical testing of a new form ofimmunotherapy. Suppressor cell lines can be useful for enhancingallograft tolerance induction or down-modulating autoimmune diseases.

The present invention comprises a method of isolating a T_(reg) cellfrom an umbilical cord blood sample. The articles “a” and “an” are usedherein to refer to one or to more than one (i.e., to at least one) ofthe grammatical object of the article. By way of example, “a T_(reg)cell” means one T_(reg) cell or more than one T_(reg) cell. The T_(reg)cell isolated using the methods of the present invention, asdemonstrated by the data disclosed herein, inhibits T cellproliferation. The method of the present invention comprises isolating apopulation of cord blood mononuclear cells from an umbilical cord bloodsample. A “population” is used herein to refer to a group of cellshaving a substantially similar phenotypic characteristic, such as beingmononuclear cells, or expressing CD25RA. Methods for isolatingmononuclear cells from a biological sample, such as a cord blood sample,are well known in the art, and include, but art not limited to, using adensity gradient centrifugation techniques so that blood components,such as plasma and erythrocytes, are separated from mononuclear cells.Methods for isolating mononuclear cells from a blood sample include theFicoll-Hypaque technique.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19 and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody. The term “antibody,” as used herein, refers to animmunoglobulin molecule which is able to specifically bind to a specificepitope on an antigen. Antibodies can be intact immunoglobulins derivedfrom natural sources or from recombinant sources and can beimmunoreactive portions of intact immunoglobulins. Antibodies aretypically tetramers of immunoglobulin molecules. The antibodies in thepresent invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, Fv, Fab andF(ab)₂, as well as single chain antibodies and humanized antibodies(Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press NY; Harlow et al., 1989, Antibodies: ALaboratory Manual, Cold Spring Harbors New York; Houston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426). By the term “specifically binds,” as used herein, is meanta compound, e.g., a protein, a nucleic acid, an antibody, and the like,which recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample.

The antibody of the present invention can be bound to a physicalsupport, such as a magnetic bead, a dynal bead, a microbead, a column,an adsorption column, and an adsorption membrane. Conjugating anantibody to a physical support is well known in the art. Id.Alternatively, an antibody conjugated to a physical support, such as amagnetic bead, can be purchased from a variety of sources, such asMilteny Biotec (Auburn, Calif.).

A variety of antibodies are useful in the present invention. As will beunderstood by one skilled in the art, any antibody that can recognizeand bind to a CD antigen of interest, such as CD25, CD4, CD34, CD8,CD14, CD19 and CD56 is useful in the present invention. Methods ofmaking an using such antibodies are well known in the art. For example,polyclonal antibodies useful in the present invention are generated byimmunizing rabbits according to standard immunological techniqueswell-known in the art (see, e.g., Harlow et al., 1988, supra). Suchtechniques include immunizing an animal with a chimeric proteincomprising a portion of another protein such as a maltose bindingprotein or glutathione (GSH) tag polypeptide portion, and/or a moietysuch that the marker protein is rendered immunogenic (e.g., a markerprotein conjugated with keyhole limpet hemocyanin, KLH) and a portioncomprising the respective marker protein amino acid residues. Thechimeric proteins are produced by cloning the appropriate nucleic acidsencoding the marker protein into a plasmid vector suitable for thispurpose, such as but not limited to, pMAL-2 or pCMX.

However, the invention should not be construed as being limited solelyto methods and compositions including these antibodies or to theseportions of the protein antigens. Rather, the invention should beconstrued to include other antibodies, as that term is defined elsewhereherein, to T_(reg) cell surface marker proteins, or portions thereof.Further, the present invention should be construed to encompassantibodies, inter alia, bind to the T_(reg) proteins and they are ableto bind the protein present on Western blots, in solution in enzymelinked immunoassays, in FACS assays, in magnetic-actived cell sorting(MACS) assays, and in immunofluorescence microscopy.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibody can specifically bind with anyportion of the marker protein and the full-length protein can be used togenerate antibodies specific therefor. However, the present invention isnot limited to using the full-length protein as an immunogen. Rather,the present invention includes using an immunogenic portion of theprotein to produce an antibody that specifically binds with a specificT_(reg) cell surface protein. That is, the invention includes immunizingan animal using an immunogenic portion, or antigenic determinant, of thecell surface marker protein.

The antibodies can be produced by immunizing an animal such as, but notlimited to, a rabbit, a mouse or a goat, with a protein of theinvention, or a portion thereof, by immunizing an animal using a proteincomprising at least a portion of an T_(reg) cell surface antigen, or afusion protein including a tag polypeptide portion comprising, forexample, a maltose binding protein tag polypeptide portion, covalentlylinked with a portion comprising the appropriate amino acid residues.One skilled in the art would appreciate, based upon the disclosureprovided herein, that smaller fragments of these proteins can also beused to produce antibodies that specifically bind an T_(reg) cellsurface protein.

The invention encompasses polyclonal, monoclonal, synthetic antibodies,and the like. The present invention further comprises the use ofbiologically active fragments of antibodies, such as an Fab fragment, anF(ab′)₂ fragment, an Fv fragment and an scFv fragment of an antibody.One skilled in the art would understand, based upon the disclosureprovided herein, that the crucial feature of the antibody of theinvention is that the antibody bind specifically with an T_(reg) cellsurface protein. That is, the antibody of the invention recognizes anT_(reg) cell or a fragment thereof (e.g., an immunogenic portion orantigenic determinant thereof), on Western blots, in immunostaining ofcells, or immunoprecipitates the protein using standard methodswell-known in the art.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibodies can be used to immunoprecipitateand/or immuno-affinity purify their cognate antigen as described indetail elsewhere herein, and additionally, by using methods well-knownin the art. In addition, the antibody can be used to isolate a T_(reg)in a population of cord blood mononuclear cells derived from anumbilical cord blood sample. Thus, by using an antibody to a T_(reg)cell surface marker, such as CD25, a T_(reg) can be identified, enrichedor isolated. One skilled in the art would understand, based upon thedisclosure provided herein, that any marker, either native orgenetically engineered, expressed on an T_(reg) cell surface, is thususeful in the present invention.

The skilled artisan would appreciate, based upon the disclosure providedherein, that that present invention includes use of either a singleantibody recognizing a single T_(reg) antigen but that the invention isnot limited to use of a single antibody. Instead, the inventionencompasses use of at least one antibody where the antibodies can bedirected to the same or different T_(reg) antigens.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, supra).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, Id.) and in Tuszynski et al. (1988, Blood,72:109-115). Quantities of the desired peptide may also be synthesizedusing chemical synthesis technology. Alternatively, DNA encoding thedesired peptide may be cloned and expressed from an appropriate promotersequence in cells suitable for the generation of large quantities ofpeptide. Monoclonal antibodies directed against the peptide aregenerated from mice immunized with the peptide using standard proceduresas referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody of the invention may be “humanized”using the technology described in, for example, Wright et al., id., andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst, 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

Once expressed, whole antibodies, dimers derived therefrom, individuallight and heavy chains, or other forms of antibodies can be purifiedaccording to standard procedures known in the art.

In one embodiment of the invention, a phasge antibody library may begenerated. To generate a phage antibody library, a cDNA library is firstobtained from mRNA which is isolated from cells, e.g., the hybridoma,which express the desired protein to be expressed on the phage surface,e.g., the desired antibody. cDNA copies of the mRNA are produced usingreverse transcriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al., supra.

Bacteriophage which encode the desired antibody, may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed, such as anT_(reg) cell surface antigen. Thus, when bacteriophage which express aspecific antibody are incubated in the presence of a cell whichexpresses the corresponding antigen, the bacteriophage will bind to thecell. Bacteriophage which do not express the antibody will not bind tothe cell.

The antibodies used in the present invention are placed in contact withthe population of cord blood mononuclear cells isolated from a humanumbilical cord sample under conditions suitable for formation of a cordblood mononuclear cell-antibody complex.

That is, the method of the present invention comprises contacting apopulation of cord blood mononuclear cells with an antibody thatspecifically binds to a T_(reg) cell in the population of cord bloodmononuclear cells so that the antibody binds to an antigen, includingCD25, on the T_(reg) cell. “Conditions suitable” is used herein to referto temperature, pH, buffers, time, and other factors that facilitate thebinding of an antibody to its cognate antigen or to a cell, such as aT_(reg) cell. Conditions that are suitable for an antibody binding to anantigen or to a cell, or to an antigen on a cell, are well known in theart and are usually from about 4° C. to about 20° C. to about 37° C. fora period of time from about 2 minutes to about 5 minutes to about 30minutes to about 1 hour to about 24 hours. Various buffers are wellknown in the art and include, for example, Tris, phosphate bufferedsaline, and the like. Various examples of conditions suitable for theformation of a cord blood mononuclear cell-antibody complex aredescribed in, for example, Harlow et al., 1988, supra.

The present invention further comprises the step of substantiallyseparating the cord blood mononuclear cell-antibody complex from apopulation of cord blood mononuclear cells. That is, as demonstrated bythe data disclosed herein, the present method of isolating a T_(reg)cell that inhibits T cell proliferation from an umbilical cord bloodsample comprises substantially separating such a T_(reg) cell from othercord blood mononuclear cells in a sample. As the term is used herein,“substantially separated from” or “substantially separating” refers tothe characteristic of a population of first substances being removedfrom the proximity of a population of second substances, wherein thepopulation of first substances is not necessarily devoid of the secondsubstance, and the population of second substances is not necessarilydevoid of the first substance. However, a population of first substancesthat is “substantially separated from” a population of second substanceshas a measurably lower content of second substances as compared to thenon-separated mixture of first and second substances.

Various techniques may be employed to separate a T_(reg) bound to anantibody, such as an anti-CD25 antibody, from cells that do not have anantibody bound cell surface marker by removing antibody-bound T_(reg)cells from the cell mixture of cord blood mononuclear cells.Alternatively, various techniques may be employed to separate theT_(reg) containing an antibody-bound cell surface marker from cells thatdo not have an antibody bound cell surface marker by removing from thecell mixture T_(reg) not bound by an antibody.

In one embodiment, the CD25 cell surface marker is used to separateantibody-bound T_(reg) from T cells and other cord blood mononuclearcells not conjugated with antibody. In one aspect of the invention, theantibodies may be attached to a solid support to allow for crudeseparation. The separation techniques employed should maximize theretention of viability of the fraction to be collected. For “relativelycrude” separations, that is, separations where up to 10%, usually notmore than about 5%, preferably not more than about 1%, of the totalcells present having the marker, may remain with the cell population tobe retained, various techniques of different efficacy may be employed.The particular technique employed will depend upon efficiency ofseparation, cytotoxicity of the methodology, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill, all of which is within the ability of the ordinary skilledartisan.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads or dynal beads, affinity chromatography,cytotoxic agents joined to a monoclonal antibody or used in conjunctionwith a monoclonal antibody, e.g., complement and cytotoxins, and“panning” with antibody attached to a solid matrix, e.g., plate, orother convenient technique. Techniques providing accurate separationinclude fluorescence activated cell sorters, which can have varyingdegrees of sophistication, e.g., a plurality of color channels, lowangle and obtuse light scattering detecting channels, impedancechannels, etc., as well as magnetic activated cell sorters.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation, biotin, which can beremoved with avidin or streptavidin bound to a support, fluorochromes,such as FITC, which can be used with a fluorescence activated cellsorter, or the like, to allow for ease of separation of the particularcell type. Any technique may be employed which is not unduly detrimentalto the viability of the remaining cells. Other techniques include, butare not limited to, dense particles for density centrifugation, anadsorption column, an adsorption membrane, and the like.

In one embodiment of the invention, an antibody specific for an cordblood derived cell surface marker is conjugated to a magnetic bead. Apopulation of cord blood derived mononuclear cells is contacted with themagnetic bead-antibody conjugate, under conditions suitable for bindingof the antibody conjugate to an T_(reg) cell surface antigen, such asCD25, displaying the antigen. After incubation under conditions suitablefor binding, such as, but not limited to, an incubation at 4° C. for 20minutes, a T_(reg) positive for the antigen are selected by passing theentire sample through a magnetic-based separation apparatus. Uponevacuation or elution of free solution from the apparatus, only themagnetically-retained marker-containing cells will remain. Theantigen-containing T_(reg) cells are then eluted from the apparatus,resulting in an enriched, isolated or purified population of T_(reg)cells. In one aspect of the invention, a T_(reg) marker is CD25.

After substantial isolation of the cells lacking comprising a T_(reg)antigen, such as CD25, generally by at least about 50%, preferably atleast about 70%, even more preferably about 80%, even more preferablyabout 90% or greater than 90%, the cells can be separated by afluorescence activated cell sorter or other methodology having highspecificity, such as magnetic activated cell sorting (MACS). Multi-coloranalyses can be employed with the FACS which is particularly convenient.

In order to increase the stringency of the of the present method ofisolating a T_(reg) cell from a human umbilical cord blood sample, aT_(reg) cell that have been substantially separated from a population ofcord blood mononuclear cells can be contacted again with an antibodythat specifically binds CD25 under conditions suitable for formation ofa cord blood mononuclear cell-antibody complex followed by substantiallyseparating the cord blood mononuclear cell-antibody complex from thepopulation of cord blood mononuclear cells. This step can be performedone or more times to isolate a human T_(reg) cell from an umbilical cordblood sample.

The present invention further comprises a method of multiplying,expanding or otherwise culturing a T_(reg) cell isolated using themethods disclosed herein. As demonstrated by the data disclosed herein,multiplying a T_(reg) cell isolated by the methods of the presentinvention can by multiplied by about 100 fold using the methodsdisclosed herein. Following isolation, a T_(reg) cell is incubated incell medium in a culture apparatus for a period of time or until thecells reach confluency before passing the cells to another cultureapparatus. The culturing apparatus can be of any culture apparatuscommonly used in culturing cells in vitro. Preferably, the level ofconfluence is greater than 70% before passing the cells to anotherculture apparatus. More preferably, the level of confluence is greaterthan 90%. A period of time can be any time suitable for the culture ofcells in vitro. T_(reg) cell medium may be replaced during the cultureof the T_(reg) cells at any time. Preferably, the T_(reg) cell medium isreplaced every 3 to 4 days. T_(reg) cells are then harvested from theculture apparatus whereupon the T_(reg) cells can be used immediately orcryopreserved to be stored for use at a later time. T_(reg) cells may beharvested by trypsinization, EDTA treatment, or any other procedure usedto harvest cells from a culture apparatus.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

The medium used to multiply the T_(reg) cells of the present inventioncomprises an antibody to CD3, and antibody to CD28, and a cytokine,preferably, but not limited to, IL-2. This is because, as demonstratedby the data disclosed herein, a cell isolated by the methods of thepresent invention can be multiplied approximately 100 fold by culturingthe cell with an antibody that binds CD3, and antibody that binds CD28,and IL-2. Further, as also disclosed herein, cells multiplied using themethods of the present invention are uniform and potent suppressorcells. Further, since the T_(reg) cells of the present invention areimmunologically naive, such cells can be administered to an animal,preferably a mammal, even more preferably a human, to suppress an immunereaction, such as those common to autoimmune diseases such as diabetes,psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhancingallograft tolerance induction, transplant rejection, and the like. Inaddition, the cells of the present invention can be used for thetreatment of any condition in which a diminished or otherwise inhibitedimmune response, especially a cell-mediated immune response, isdesirable to treat or alleviate the disease.

The CD3 antibody and CD28 antibody used in the methods of the presentinvention can be any of the antibodies known in the art, those disclosedelsewhere herein, or those yet to be discovered. Further, andpreferably, the antibodies of the present invention are conjugated orotherwise attached to a bead, such as a magnetic bead or a dynal bead.Such beads are known in the art and are described elsewhere herein.

The medium of the present invention further comprises a cytokine,preferably IL-2. This is because, as demonstrated by the data disclosedherein, the addition of IL-2 to the medium used in the methods of thepresent invention results in an approximate 100 fold expansion of theT_(reg) cells of the present invention. IL-2 and other cytokines arewell known in the art and are available commercially from varioussources.

The T_(reg) of the present invention further comprises certain antigenicmarkers, some of which are present when a T_(reg) cell is isolated froman umbilical cord blood sample, some of which are present when theT_(reg) cell is multiplied, cultured, or otherwise expanded according tothe methods of the present invention. Such antigenic markers are usefulin the identification of a T_(reg) cell of the present invention, andallow one of skill in the art to determine if a T_(reg) cell isolatedand multiplied according to the methods of the present invention has theproperties and biological activities of a T_(reg) cell of the presentinvention. Such biological activities include, but are not limited to,suppression of an allogeneic immune response, inhibition of cytokineaccumulation in an immune response accompanied by less inhibition ofchemokine production, the production of IL-2, IL-10 and gammainterferon, the expression of TGF-beta latency associated protein (LAP),and suppressor activity independent of IL-10 and TGF-beta. Markers onthe T_(reg) cell of the present invention include, but are not limitedto, CD25, CD4, CTLA4, CD27, CD26L and Fox P3.

The present invention further comprises a method for inhibitingproliferation of a T cell. Such inhibition can occur in vitro or invivo, preferably in an animal, more preferably in a mammal, even morepreferably in a human. This is because, as demonstrated by the datadisclosed herein, HLA mismatched T cells in a mixed lymphocyte reaction(MLR) were inhibited from proliferating by a factor greater than about95% in the presence of a T_(reg) cell isolated and multiplied accordingto the methods of the present invention. Further, as demonstrated by thedata disclosed herein, T_(reg) cells isolated and/or multipliedaccording to the methods of the present invention are potent suppressorsof T cell proliferation at ratios of from about 1:16 to about 1:32(T_(reg):T cell). Further, the T_(reg) cells of the present inventionare active in suppressing an immune response when a antigen presentingcell, such as a dendritic cell, is mature and activated. Thus, the cellsof the present invention can be used to inhibit active immune responsesor to prevent an immune response.

The method of the present invention comprises contacting a T cell with aT_(reg) cell isolated and/or expanded according to the methods of thepresent invention such that the proliferation of a T cell is inhibited.The T_(reg) cell can be administered using techniques well known in thatart so that a T_(reg) contacts, or is in proximity, to an immune cell,such as a T cell, dendritic cell, plasma cell, and the like.

The method of inhibiting the proliferation of a T cell using a T_(reg)isolated and/or cultured according to the methods of the presentinvention encompasses the preparation and use of pharmaceuticalcompositions comprising a T_(reg) of the invention as an activeingredient. Such a pharmaceutical composition may consist of the activeingredient alone, as a combination of at least one active ingredient(e.g., an effective dose of an T_(reg)) in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient and one or more pharmaceuticallyacceptable carriers, one or more additional (active and/or inactive)ingredients, or some combination of these.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs, birds including commercially relevant birds suchas chickens, ducks, geese, and turkeys, fish including farm-raised fishand aquarium fish, and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,intra-lesional, buccal, ophthalmic, intravenous, intra-organ or anotherroute of administration. Other contemplated formulations includeprojected nanoparticles, liposomal preparations, resealed erythrocytescontaining the active ingredient, and immunologically-basedformulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers and other immune inhibitory compounds, such as cyclosporine,steroids, antibodies to pro-inflammatory cytokines, inhibitorycytokines, such as IL-10, and the like.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non toxic parenterally acceptable diluent or solvent,such as water or 1,3 butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as synthetic monoor di-glycerides. Other parentally-administrable formulations which areuseful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

The T_(reg) of the invention and/or a T_(reg) expanded using the methodsof the present invention, can be administered to an animal, preferably ahuman. When the T_(reg) of the invention are administered, the amount ofcells administered can range from about 100,000 cells to about 300billion cells wherein the cells are infused into the animal, preferably,a human patient in need thereof. While the precise dosage administeredwill vary depending upon any number of factors, including but notlimited to, the type of animal and type of disease state being treated,the age of the animal and the route of administration.

The T_(reg) may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

An T_(reg) may be co-administered with the various other compounds(cytokines, chemotherapeutic drugs, immunosuppressive drugs, among manyothers). Alternatively, the compound(s) may be administered an hour, aday, a week, a month, or even more, in advance of the T_(reg,) or anypermutation thereof. Further, the compound(s) may be administered anhour, a day, a week, or even more, after administration of a T_(reg), orany permutation thereof. The frequency and administration regimen willbe readily apparent to the skilled artisan and will depend upon anynumber of factors such as, but not limited to, the type and severity ofthe disease being treated, the age and health status of the animal, theidentity of the compound or compounds being administered, the route ofadministration of the various compounds and the T_(reg), and the like.

Further, it would be appreciated by one skilled in the art, based uponthe disclosure provided herein, that where the T_(reg) is to beadministered to a mammal, the cells can be treated so that they are in a“state of no growth”; that is, the cells are incapable of dividing whenadministered to a mammal. As disclosed elsewhere herein, the cells canbe irradiated to render them incapable of growth or division onceadministered into a mammal. Other methods, including haptenization(e.g., using dinitrophenyl and other compounds), are known in the artfor rendering cells to be administered, especially to a human, incapableof growth.

The invention includes various kits which comprise the reagents used toisolate a T_(reg) from a human umbilical cord blood sample. The kitcomprises an antibody that specifically binds to a molecule on thesurface of a T_(reg) cell, such as CD25, an applicator, andinstructional materials which describe use of the kit to perform themethods of the invention. Although exemplary kits are described below,the contents of other useful kits will be apparent to the skilledartisan in light of the present disclosure. Each of these kits isincluded within the invention. By the term “applicator,” as the term isused herein, is meant any device including, but not limited to, ahypodermic syringe, a pipette, and the like, for administering thecompounds and compositions of the invention.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in the kit for effecting alleviating ortreating the various diseases or disorders recited herein. Optionally,or alternately, the instructional material may describe one or moremethods of alleviating the diseases or disorders in a cell or a tissueor a mammal, including as disclosed elsewhere herein.

The invention further includes a kit for multiplying a T_(reg) from ahuman umbilical cord blood sample. The kit comprises an antibody thatspecifically binds to CD3 and an antibody that specifically binds toCD28. The antibodies of the present kit can be isolated antibodies,antibodies bound to a physical support, such as a magnetic bead, orother antibodies described elsewhere herein or known in the art. The kitcan further comprise a cytokine, such as IL-2, for culturing,multiplying or otherwise expanding a T_(reg) of the present invention.The kit is used pursuant to the methods disclosed in the invention. Thekit can further comprise an applicator and an instructional material forthe use thereof.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLES Example 1 Isolation and Culture of CD45RA⁺ Cells From AdultHuman Peripheral Blood

MACS Purification of CD25⁺ and CD25⁻ Cells for Culture

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coatpreparations, which were derived from the whole blood of normal healthyvolunteer donors (Memorial Blood Centers, Minneapolis, Minn.). Leukocyterich buffy coat cells were centrifuged over Ficoll-Hypaque layers tocollect PBMC. CD25⁺ cells were isolated using the following indirectantibody based microbeads. PBMC were stained with anti-CD25-FITC, clone2A3 (Becton Dickinson Immunocytometry Systems, San Jose, Calif.), washedand then bound secondarily to anti-FITC multi-sort microbeads (5microliters/10⁷ cells, Miltenyi Biotec, Auburn, Calif.) and positivelyselected. The CD25⁺ cells were reapplied to a second column, washed andre-eluted. After column purification, the anti-FITC multisort beads weredetached. The CD25⁺ cells were further depleted of CD8, CD14, CD19,CD20, and CD56 expressing cells with a cocktail of mAb-coated microbeadsfor lineage depletion. These CD25⁺ lineage depleted cells (DC-8×-minus)were then selected for CD45RA by direct positive selection withanti-CD45RA microbeads (20 microliters/10⁷ cells, Miltenyi). In somecases, a further purification of anti-HLA-DR+ cells was isolated fromthe CD45RA⁻ cells by positive selection with anti-HLA-DR microbeads (20microliters/10⁷ cells, Miltenyi). The CD25⁻ cells were further depletedof CD25 by a second round of depletion with direct anti-CD25 microbeads(20 microliters/10⁷ cells, Miltenyi). After CD25 depletion these cellswere then positively selected for CD4 with direct anti-CD4 microbeads(20 microliters/10⁷ cells, Miltenyi).

Culture of CD25⁺ and CD25⁻ Cells

Isolated CD25⁺ cells, CD25⁺ subsets, or CD4⁺CD25⁻ control cells werecultured at 1 million total cells/ml in 24 well plates. An equal numberof irradiated CD4⁺CD25⁻ feeder cells were added to the cultures.Anti-CD3/CD28 mAb-coated dynabeads (University of Pennsylvania,Philadelphia, Pa.) were added at a 3:1 bead to cell ratio as a growthstimulus, and moderate dose IL-2 was added on day 3, at 50 IU/ml in thefresh media (Chiron, Emeryville, Calif.) (Godfrey, et. al., 2004, Blood,104: 453-461; Levine, et al., 1998, J. Hematother., 7: 437-48). For cordblood cultures, feeder cells were not required for culture orpreservation of suppressor function, and therefore were not used. Cellcultures were split as needed, approximately ⅓ every 3 days during thefast growth phase. Culture media was RPMI-1640 (Invitrogen-Gibco,Carlsbad, Calif.) supplemented with 10% FCS (Invitrogen-Gibco), andL-glutamine, penicillin, and streptomycin.

Cell lines previously isolated using a MACS based strategy for isolationof CD4 ⁺CD25⁺ cells for T_(reg) cell line generation from adult bloodexhibited variable potency, which was possibly caused by contaminatingconventional T cells. To improve on the purification strategy, CD25⁺cells were analyzed for subsets by flow cytometric analysis. A subset ofCD4⁺CD25⁺ cells could be further enriched for suppressor cells, orconversely, a subset might be enriched for conventional T cells. Theapproach described herein was to characterize subset markers,selectively purify these cell subsets, and then evaluate their abilityto form cell lines with potent suppressor function.

The first markers analyzed were CD45RO, a marker of memory cells, andCD45RA, a marker of naive T cells. The CD45RO⁺ subset of CD4⁺CD25⁺ cellshas been reported to contain the suppressor cells, as freshly isolatedCD4⁺CD25⁺CD45RO⁺ cells contained most of the suppressor activity ofCD4⁺CD25⁺ cells (Jonuleit, et al., 2001, J. Exp. Med., 193: 1285-1294).CD4⁺CD25⁺CD45RO-minus cells had minimal suppressor activity infunctional assays (secondary allogeneic MLR). The CD4⁺CD25⁺CD45RO-minuscells were suggested to be conventional T cells. As conventional T cellstypically overgrow suppressor cells in culture, depletion of theCD4⁺CD25⁺CD45RO-minus cells was investigated as a means to improve cellline generation.

Purified CD4⁺CD25⁺ cells were assessed for CD45RA and CD45RO expressionby FACS analysis (FIG. 1). The CD25⁺ cells coexpressed CD45RO onapproximately 80% the cells (mean 80%, range 62-89, n=6). The CD25⁺cells also contained a subset of cells which coexpressed CD45RA onapproximately 20% of cells (mean 24%, range 15-38, n=6). High levelexpression of these antigens was mutually exclusive, but there are asignificant number of double positive cells only expressing low amountsof both antigens, and a two-color dot-plot reveals a whole spectrum ofexpression. The CD45RA⁺ cells did not express CD25 at the highestlevels, and the CD25 bright cells, thought to be the true T_(reg) cells,appear to not express CD45RA (FIG. 2). These data therefore indicatethat depletion of the CD45RA subset would further enrich for the CD45RO⁺T_(reg) cells, and elimination of these CD4⁺CD25⁺CD45RA⁺ cells wasattempted.

The indirect purification system with a cleavable microbead allowedpurification of CD25⁺ cells, removal of the magnetic bead, and thendepletion of the CD45RA⁺ cells. Using a typical buffy coat, the startingmaterial was approximately 500 million cells, and through purificationobtain approximately 10 million CD25⁺ cells, 7 million CD25⁺lineage-negative cells, 5 million CD45RA-minus cells, and 1 millionCD45RA⁺ cells were derived. Purifications from two donors are depicted(FIG. 1). These various CD25⁺ populations were cultured with one roundof stimulation with anti-CD3/28 beads. After 3 weeks, the cell lineswere tested for ability to suppress allo MLR.

To evaluate suppressor function of these cell lines, an HLA-mismatchedallo-MLR assay was used as a functional readout. The CD4⁺ responder andDC stimulated MLR assays are very robust and consistent amongst donors,and therefore served as our standard measure of suppression. All celllines were initially screened for suppressor activity in MLR after 2-3weeks of culture, and then further analyzed over the next 3-4 weeks.

Surprisingly, it was the CD45RA⁺ derived cell lines (which were expectedto be conventional T cells and lack activity), that demonstrated potentsuppressor activity (FIG. 11). In contrast, the CD45RA-minus cellderived lines had very poor suppressor function. Twelve cell line donorswere tested, and 10/12 RA cell lines demonstrated potent suppressoractivity and 2/12 RA-minus lines demonstrated similar activity. Thesedata, and the percent suppression mediated by these cell lines isdepicted in a scatter plot, which also includes straight CD25⁺ selectedcells from adult blood and cord blood (FIG. 3).

Two Main Subsets of Human CD25⁺ Cells, CD45RA⁺ and HLA-DR⁺

Further analysis of CD25⁺ cell subsets revealed that the CD45RA⁺ cellswere distinct from the CD25⁺ bright, classically described human T_(reg)cell (Baecher-Allan, et al., 2001, J. Immunol., 167: 1245-1253; Godfrey,et. al., 2004, Blood 104: 453-461; Hoffmann, et al., 2004, Blood 104:895-903). Prior reports have noted these cells to be CD25⁺ hi, andCTLA-4 positive (Jonuleit, et al., 2001, J. Exp. Med., 193: 1285-1294).The data disclosed herein demonstrate that the CD45RA cells aredistinct. In two color dot plots, the CD45RA cells are shown to beintracellular CTLA-4 negative (FIG. 4), HLADR negative (FIG. 4). Doublestaining for CD25 verus HLA-DR reveals that the HLA-DR⁺ cells are theCD25⁺ bright cells (FIG. 4), and they double stain for intracellularCTLA-4 (FIG. 4). This demonstrates two distinct cell subsets.

Purification of the HLA-DR cells was performed by further positiveselection of the CD45RA(−) cell population. This left three subsets ofCD25⁺ cells, the CD45RA⁺ cells, the HLA-DR⁺ cells, and the “doublenegatives”. All three subsets were cultured and tested for suppressorfunction. The CD45RA⁺ derived cell lines mediated the most potentsuppressor function. The double negative cells mediated a middle levelof suppression and the HLA-DR⁺ mediated the worst suppressive activity(FIG. 5).

Example 2 Adult Human CD45RA⁺ Cells Suppress T-Cell Proliferation

Responder and Stimulator Cells for Mixed Lymphocyte Reaction (MLR) AssayCultures

CD4⁺CD25⁻ responder T-cells were isolated from buffy coat preparationsderived from the whole blood of normal healthy volunteer donors(Memorial Blood Centers, Minneapolis, Minn.). Cells were centrifugedover Ficoll-Hypaque to collect PBMC. PBMC were first depleted of CD25⁺cells with anti-CD25 mAb-coated microbeads (Miltenyi-Biotec), beforeCD4⁺ T-cells were isolated by positive selection with anti-CD4mAb-coated magnetic microbeads (Miltenyi-Biotec). Cells were routinely96-98% pure by FACS analysis. Immature dendritic cells (DC) weregenerated from CD14⁺ monocytes (Sallusto and Lanzavecchia, 1994, J. Exp.Med., 179: 1109-1118), isolated from PBMC, by magnetic bead basedpurification (Miltenyi-Biotec), and were cultured in X-vivo-15(BioWhittaker, Walkersville, Md.) media at 1 million cells/mlsupplemented with GM-CSF (50 ng/ml final) and IL-4 (20 ng/ml final; R&DSystems, Minneapolis, Minn.). Cells were cultured for 5-10 days beforeuse as stimulators in MLR. For some experiments, DC were matured withLPS (Sigma, St. Louis, Mo.) (100 ng/ml), or TNF-alpha (20 ng/ml final)and Poly I:C, a Toll-like receptor (TLR)-3 agonist ligand (20 μg/mlfinal) (Sigma) for two days (Godfrey, et al., 2004, Blood 103:1158-1165; Cella, et al., 1999, J. Exp. Med., 189: 821-829). DCstimulators were irradiated at 30 Gy.

MLR Assay Culture

5×10⁴ responding CD4⁺CD25⁻ T-cells and 5×10³ DC stimulator antigenpresenting cells (APC) were cultured per well in 96 well U-bottomplates. Cultured suppressor or conventional T-cell lines were added at2.5×10⁴ per well for standard assays, or in graded numbers for titrationexperiments. For antibody blocking experiments, 10⁴, or 5×10³ suppressorcells were used. Culture media was RPMI-1640 supplemented with 10% FCS.Wells were pulsed on days 3, 5, 6, and 7 with ³H-thymidine for the last16 hours of culture. Each timepoint had 6 replicates. Results expressedin counts per minute. Data was collected with a direct beta counter (noliquid scintillation), thus the magnitude of the results was lower butproportionally correct.

Cytokine Analysis

MLR culture supernatants were spun free of cells and aliquots werefrozen at −80° C. For re-stimulations, anti-CD3/CD28 beads were used ata 1:1 bead to cell ratio. Supernatants were evaluated by the Luminexassay system with a latex bead-based multianalyte system (R&D Systems,Minneapolis, Minn.).

Potency of Suppression Mediated by CD45RA⁺ Derived Cell Lines

Titration experiments were performed to further evaluate suppressionpotency. Lowering the number of suppressors added to standard DCstimulated MLR cultures demonstrated that nearly full inhibitoryactivity of cord blood derived suppressors was maintained out to a ratioof 1:16 or 1:32 (as few as 1500 suppressors to 50,000 responders). Thisis slightly more potent (˜2-fold) than the selected most potent adultderived cell lines (CD25⁺ lineage⁻).

As a further assessment of potency, suppressor cell lines were evaluatedin MLR where the DC stimulators had been matured. Activation/maturationof DC with lipo-polysaccharide (LPS), (TLR4 ligand), or the combinationof TNF/polyIC (double stranded RNA analog-TLR 3 ligand), did not lead tobypass of suppression. In addition, inclusion of LPS or TNF/Poly IC inthe MLR culture, also did not bypass suppression. Thus the CD45RA⁺derived T_(reg) cells were both potent and activated DC, whichabundantly express costimulatory molecules and cytokines. Further, DCand the expressed costimulatory molecules were not able to bypass thesuppressive effect of the CD4⁺CD25⁺ cells.

CD45RA⁺ Derived Suppressor Cells Impair Cytokine Production in MLR

To determine the effects of the suppressor cell lines on responder Tcells in MLR assays, the culture supernatants were evaluated for thepresence and magnitude of multiple cytokines. Suppressor cell additionto MLR assays markedly reduced accumulation of T cell activationdependent cytokines, including IL-2, IFN-gamma, GM-CSF, TNF-α, andIL-10. Activation dependent cytokine accumulation was minimal at alltimepoints during the suppressed MLR. These data demonstrate a markedimpairment of T cell activation.

Reactivation of Suppressor Cell Lines Induces Minimal CytokineProduction

To determine the functional capabilities of the suppressive versusconventional T-cell lines we evaluated their potential for cytokineproduction and cell surface molecule expression after re-stimulation.Cell lines were re-stimulated with anti-CD3/CD28 beads for potentreactivation, and supernatants harvested at defined timepoints foranalysis of cytokine content by luminex bead based assay. The CD45RA⁺derived cell lines produced essentially no IL-2, IFN-γ, or IL-10, whilecontrol CD25⁻ derived cell lines produced high levels of thesecytokines. The accumulation of TNF, GM-CSF, and IL-5, and the chemokineIL-8, was also markedly reduced as compared to control cell lines.

Example 3 Isolation and Culture of Human Cord Blood Having a CD45RA⁺Phenotype

The critical role of T_(reg) cells in cord blood immunology andtransplantation has not been generally appreciated (Barker, et al.,2003, Crit. Rev. Oncol. Hematol., 48: 35-43). Many researchers use totalCD4⁺ selected cord blood populations as a representative model of trulynaive T cells for purposes of immunological characterization (Kaminski,et al., 2003, Blood 102: 4608-4617). However, in light of the datadisclosed herein, some prior studies may need to be re-evaluated withCD25 depleted CD4⁺ cells (Jonuleit, et al., 2000, J. Exp. Med., 192:1213-1222). These cells may be a contributing factor to the low rate ofGVHD experienced in cord blood transplantation (CBT) (Wadlow, et al.,2002, Biol. Blood. Marrow. Transplant., 8: 637-647).

MACS Purification of CD25⁺ and CD25⁻CD4⁺ T-Cells

CD25⁺ and CD25⁻CD4⁺ T-cells were isolated from umbilical cord blood (RedCross, Saint Paul, Minn.). Cord blood mononuclear cells were prepared bycentrifugation over Ficoll-Hypaque according to the manufacturer'sdirections. After CD34⁺ depletion with magnetic microbeads (MiltenyiBiotec, Auburn, Calif.), CD25⁺ cells were isolated by positive selectionwith directly conjugated anti-CD25 magnetic microbeads (4 microlitersper 10⁷ cells; Miltenyi Biotec). Cells were then applied to a secondmagnetic column, washed, and re-eluted. After the double columnprocedure cells were routinely >90% pure (for CD4/CD25) by FACSanalysis. The non-CD25 fraction was then applied to another magneticcolumn to deplete any remaining CD25⁺ cells, before isolation ofCD4⁺CD25⁻ cells by positive selection with anti-CD4 mAb-coatedmicrobeads (Miltenyi Biotec). Stringent purification of adult CD25⁺cells used anti-CD25-FITC and anti-FITC microbeads (2 microliters per10⁷ cells), and passage over magnetic column and elution for two cycles.This was followed by releasing the magnetic beads, and subsequentlineage depletion with anti-CD8, CD14, CD19, and CD56 direct conjugatedmicrobeads (CD4⁺CD25⁺⁺lin-) as described in Godfrey, et al. (2004, Blood104: 453-461).

Culture of Cord Blood T_(reg) Cells

Isolated CD4⁺CD25⁺ cells or control CD4⁺CD25⁻ cells were cultured asdescribed in Godfrey, et al. (2004, Blood 104: 453-461) withanti-CD3/CD28 mAb-coated dynabeads (obtained from the University ofPennsylvania and described in Levine, et al., 1998, Hematother.,7:437-448) at a three-to-one bead to cell ratio. Cells were cultured at1 million total cells/ml in 24 well plates. IL-2 was added on day 3 at50 IU/ml (Chiron, Emeryville, Calif.). In contrast to the prior study,all lines were cultured without feeder cells. For cord blood cultures,the addition of irradiated feeder cells (CD4⁺CD25⁻ cells) (Godfrey, etal. 2004, Blood 104: 453-461), was neither helpful for expansion, norpreservation of suppressor function, therefore feeders were omitted tosimplify the culture protocol. Cell cultures were split as needed,approximately one-to-three every three days during the fast growthphase. Culture media was RPMI-1640 (Invitrogen-Gibco, Carlsbad, Calif.)supplemented with 10% fetal calf serum (FCS; Invitrogen-Gibco), andL-glutamine, penicillin, and streptomycin.

Cord Blood Contains a Distinct Population of CD4⁺CD25⁺ Cells

Cord blood mononuclear cells (CBMC) have been shown to contain CD4⁺T-cells that co-express CD25. Therefore, the present results disclose anevaluation of the potential of these cells for suppressor functions andsuppressor cell line generations. FACS analysis was used to confirmedthat CBMC contained a significant population of CD4⁺ T-cells thatco-express CD25⁺, and that this was a discrete population of cells (FIG.6A). In contrast, adult blood contains CD4⁺ T-cells with a broadspectrum of levels of CD25, including a large population ofnon-suppressive CD25-dim cells (FIG. 6B). Approximately 5% of the cordblood CD4⁺ T-cells distinctly expressed CD25⁺ (mean 5.2%, range2.3-9.5%, n=20), a slightly higher percentage than present in adultperipheral blood CD4⁺ T cells (mean 3.8% of CD4⁺). For direct comparisonof purification, CD25⁺ T_(reg) cells were isolated from both adult andcord blood using an identical MACS based protocol (direct anti-CD25based selection). The CD25⁺ cells purified from cord blood contained amore focused population of CD25⁺ bright cells (MFI 320 vs 130), andfewer CD25-dim or CD25-negative cells (FIG. 6C), mean 9% (range 5-21%,n=10) and 3% (range 1-9%, n=10) respectively. In comparison, the CD25⁺cells derived from adult blood were found to contain more CD25-dim andCD25-negative cells (FIG. 6D), mean 30% (range 25-38%, n=10), and 10%(range 2-24%, n=10) respectively.

Cord Blood CD25⁺ Cells are CD45RA⁺

Cord blood CD25⁺ cells were purified by direct selection. They werestained for CD25 and CD45RA. The cells are predominantly CD45RA⁺ (FIG.7).

MACS Selected Cord Blood CD4⁺CD25⁺ Derived Cell Lines Consistently HavePotent Suppressor Function

Stimulating stringently purified adult derived CD25⁺ T_(reg) cells withanti-CD3/CD28 coated beads, supplemented with IL-2, induces significantexpansion. This culture strategy was used to generate cell lines frompurified adult or cord blood CD4⁺CD25⁺ cells, as well as cord bloodCD4⁺CD25⁻ cells for comparison. The cord blood derived CD4⁺CD25⁺ cellsexpanded readily in culture; approximately 100 fold over three weekswith a single initial stimulation. After three to four weeks the celllines stopped expanding in number, and were maintained in IL-2. Thus,the growth curves of these cell lines were similar to that of the adultblood derived CD4⁺CD25⁺ cell lines.

Example 4 CD45RA⁺ Cord Blood Cells Suppress T-Cell Proliferation

Responder and Stimulator Cells for Mixed Lymphocyte Reaction (MLR)Cultures

CD4⁺CD25⁻ responder T-cells were isolated from buffy coat preparationsderived from the whole blood of normal healthy volunteer donors(Memorial Blood Centers, Minneapolis, Minn.). Cells were centrifugedover Ficoll-Hypaque to collect PBMC. PBMC were first depleted of CD25⁺cells with anti-CD25 mAb-coated microbeads (Miltenyi-Biotec), beforeCD4⁺ T-cells were isolated by positive selection with anti-CD4mAb-coated magnetic microbeads (Miltenyi-Biotec). Cells were routinely96-98% pure by FACS analysis. Immature dendritic cells (DC) weregenerated from CD14⁺ monocytes (Sallusto, et al., 1994, J. Exp. Med.,179: 1109-1118), isolated from PBMC, by magnetic bead based purification(Miltenyi-Biotec), and were cultured in X-vivo-15 (BioWhittaker,Walkersville, Md.) media at 1 million cells/ml supplemented with GM-CSF(50 ng/ml final) and IL-4 (20 ng/ml final) (R&D Systems, Minneapolis,Minn.). Cells were cultured for 5-10 days before use as stimulators inMLR. For some experiments, DC were matured with LPS (Sigma, St. Louis,Mo.) (100 ng/ml), or TNF-alpha (20 ng/ml final) and Poly I:C, aToll-like receptor (TLR)-3 agonist ligand (20 μg/ml final) (Sigma) fortwo days (Spisek, et al., 2001, Cancer Immunol. Immunother., 50:417-427; Godfrey, et al., 2004, Blood 103: 1158-1165). DC stimulatorswere irradiated at 30 Gy.

MLR Assay Culture

5×10⁴ responding CD4⁺CD25⁻ T-cells and 5×10³ DC stimulator APC werecultured per well in 96 well U-bottom plates. Cultured suppressor orconventional T-cell lines were added at 2.5×10⁴ per well for standardassays, or in graded numbers for titration experiments. For antibodyblocking experiments, 10⁴ or 5×10³ suppressor cells were used. Culturemedia was RPMI-1640 supplemented with 10% FCS. Wells were pulsed on days3, 5, 6, and 7 with ³H-thymidine for the last 16 hours of culture. Eachtime-point had 6 replicates. Results were expressed in counts perminute. Data was collected with a direct beta counter (no liquidscintillation), thus the magnitude of the results was lower butproportionally correct.

Suppressor Function of T_(reg) Cells as Determined by MLR Assay

To evaluate suppressor function of the cell lines, an HLA-mismatchedallo-MLR assay was used as a functional readout. The CD4⁺ responder andDC stimulated MLR assays are very robust and consistent amongst donors,and therefore served as the standard measure of suppression. All celllines were initially screened for suppressor activity in MLR after 2-3weeks of culture, and then further analyzed over the next 3-4 weeks.Strikingly, the cell lines derived from cord blood CD25⁺ cells wereconsistently and potently suppressive (FIG. 8A). Potent suppressive celllines were isolated from 29 of 30 donors, where inhibition ofproliferation was typically >95% (FIG. 8B). Control cell lines derivedfrom adult or cord blood CD25⁻ cells were not suppressive (FIG. 8B).Cell lines derived from adult CD25⁺ cells (directly isolated),manifested weak and variable suppressive activity (FIGS. 8A and 8B). Itwas previously reported that stringent MACS based selection was requiredto generate significant, potent, suppressor cell lines in a subset ofadult donors. The suppression mediated by these lines (CD25⁺⁺lineage) isdepicted in FIGS. 8A and 8B, which demonstrates the remarkableconsistency of the cord blood derived cell lines.

To further evaluate suppression potency, titration experiments wereundertaken. Lowering the number of suppressors added to standard DCstimulated MLR cultures revealed that nearly full inhibitory activity ofcord blood derived suppressors was maintained out to a ratio of 1:16 or1:32 (as few as 1500 suppressors to 50,000 responders). This is morepotent (˜2-fold) than the selected most potent adult derived cell lines(pCD25⁺lin-) (FIG. 2C). Cell lines derived from directly selected adultCD25⁺ cells were poorly suppressive upon titration.

As a further assessment of potency, suppressor cell lines were evaluatedin MLR where the DC stimulators had been matured. Activation/maturationof DC with lipopolysaccharide (LPS), (TLR4 ligand), or the combinationof TNF/polyIC (double stranded RNA analog-TLR 3 ligand) (Cella, et al.,1999, J. Exp. Med., 189: 821-829), did not bypass suppression (FIG. 2D).In addition, inclusion of LPS or TNF/Poly IC in the MLR culture did notbypass suppression. Thus the cord derived T_(reg) cells were as potentas the best selected adult derived cell lines, and activated DC, whichabundantly express co-stimulatory molecules and cytokines, were not ableto bypass their suppressive effect.

Cord Blood Suppressor Cells Impair Cytokine Production with Less of anEffect on Chemokine Production

To determine the effects of the suppressor cell lines on responder Tcells in MLR assays, supernatants were evaluated for the presence andmagnitude of multiple cytokines. Suppressor cell addition to MLR assaysmarkedly reduced accumulation of T cell activation dependent cytokines,including IL-2, IFN-gamma, GM-CSF, TNF-α, and IL-10. Importantly, levelsof TGF-_(beta-1) accumulation did not seem to be altered. Activationdependent cytokine accumulation was minimal at all time-points duringthe suppressed MLR, with results at time of peak detection in controlDC-MLR depicted (FIG. 9A). These data demonstrate a marked impairment ofT cell activation. To determine the effects of suppression on selectedchemokine expression, MLR supernantants were evaluated for theexpression of IL-8 (CXCL8), MIP-1a (CCL3), and RANTES (CCL5),pro-inflammatory chemokines. At early time-points, no effect onaccumulation was noted (FIG. 9B). However, at late timepoints in MLR,accumulation was diminished by approximately by 50-75%. Thus, chemokineproduction appears to be less affected by suppression, and there is somedegree of selectivity in the effects of suppressor cells.

Studies of both human and mouse CD4⁺CD25⁺ T_(reg) cells have notrevealed a clear mechanism of action of in vitro suppression (Shevach,2002, Nat. Rev. Immunol., 2: 389-400). Consistent with previous studies,the cord blood derived cultured suppressor cells required cell contactfor function (did not suppress across a semi-permeable membrane), andwere not cytotoxic. Since TGF beta is variably reported to be a primaryfactor in T_(reg) mediated suppression (Chen, et al., 2003, CytokineGrowth Factor Rev., 14: 85-89), and rLAP was reported to impairsuppressor function (Nakamura, et al., 2004, J. Immunol., 172: 834-842),the TGF-beta pathway was carefully evaluated with multiple neutralizingreagents, including rLAP. Multiple antagonists of TGF-beta, even invarious combinations, were found to minimally affect suppression.Neutralizing antibodies to immunosuppressive factor IL-10 or itsreceptor had no effect on suppression. This is consistent with the lackof IL-10 production by the cord blood suppressor cells even with verystrong activating stimuli. Thus, in the presently disclosed in vitro MLRsystem, neither TGF-beta nor IL-10 appear to be the primary mediators ofsuppression, and the molecules mediating suppression remainuncharacterized.

Example 5 Characterization of Human Adult CD25RA⁺ T_(reg) Cells

Monoclonal Antibodies

To evaluate purification, cells were stained with anti-CD25-PE(cloneM-A251) (BD Pharmingen, San Diego, Calif.), which is not blocked byanti-CD25-microbeads. Other antibodies for flow cytometry includedanti-CD4-PerCP(clone SK3), from (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.); anti-CD152-PE(BNI3), anti-CD27-FITC(M-T271),anti-CD62L-APC(Dreg56), anti-CD69-FITC(FN50), anti-CD134(ACT35), from(BD Pharmingen); and anti-GITR-PE(110416), from (R&D Systems). Infunctional experiments designed for blocking suppression, neutralizingantibodies were used at 10 μg/ml. Antibodies included anti-CTLA4(BNI3)(BD Pharmingen), anti-PD1(J116) (eBioscience, San Diego, Calif.),anti-OX40(L106) from (Becton Dickinson), and anti-GITR(MAB689),anti-GITR-L(MAB6941), anti-OX40L(MAB10541), anti-IL10(MAB217),anti-IL10-Receptor-alpha(MAB274), anti-TGFbeta-1,2,3(1D11),anti-TGFbeta-1 (MAB1835), polyclonal chickenanti-TGFbeta1/1.2(AF-101-NA), from (R&D Systems).

Cultured Suppressor Cells Function Independent of IL-10 and TGF-beta

To determine if the cultured suppressor cell lines work through knownsoluble immunosuppressive or cell surface negative regulatory proteins,DC-MLR suppressor assays were treated with neutralizing or blockingmonoclonal antibodies. Assays were evaluated for the reversal ofsuppression by resumption of proliferation. Initially antibodies toIL-10, IL10R, and TGF-beta_(1,2,3) were tested alone or in variouscombinations with minimal effect. Only by combining all three antibodiesat high doses (30 μg/ml), were modest and probably non-specific reversalof suppression effects seen. Treated control cultures treated with allthree antibodies also evidenced increased proliferation, similar toT_(reg) cells treated with all three antibodies.

Flow Cytometry

For immunofluorescence staining, cells were stained for 30 minutes at 4°C. Cells were washed and run on a FACS Calibur cytometer (BectonDickinson). Data was analyzed by FlowJo software version 4.5 (Treestar,Ashland, Oreg.). Intracellular staining was performed using 2%paraformaldehyde fixed cells, followed by permeabilization and stainingin 0.1% saponin containing buffer.

Statistics

All error bars represent one standard deviation above and below themean.

Phenotype of CD45RA⁺ Derived Suppressor Lines

Cell lines were analyzed by flow cytometry for antigens associated withsuppressor cell phenotype. Suppressor cell lines were cultured inparallel with CD4⁺CD25⁻ derived cell lines which served as conventionalT-cell controls. The cell lines maintained a relatively stable phenotypeand function for the next 3-5 weeks. The CD45RA⁺ derived CD25⁺ celllines maintain remarkably uniform expression of multiple antigens,including CD25, intracellular CTLA4, CD27 and CD62L. In comparison, theCD45RA(−) derived cell lines, were heterogeneous for these antigens.

The CD45RA⁺ derived CD25⁺ cell lines maintained high expression of cellsurface CD25 and intracellular CTLA4, an expression pattern consideredtypical of the T_(reg) phenotype. This occurs despite the fact thatother activation antigens such as CD69, CD71, and OX40 have returned tobaseline expression. The CD45RA⁺ derived suppressor cell lines alsouniformly express both CD62L and CD27. Within potent adult derivedsuppressor cell lines, the cells with suppressor function reside withinthis double positive subset (Godfrey, et. al., 2004, Blood 104:453-461). CD25⁻ derived cells lines also maintain CD27 expression, butat a lower level than CD25⁺ derived cell lines. This CD27dim expressionpattern was also noted with adult derived CD25⁻ derived cell lines(Godfrey, et. al., 2004, Blood 104: 453-461).

Example 6 Characterization of Human Cord Blood Derived T_(reg) Cells

Real Time PCR

Total RNA was extracted using TRI-reagent (Molecular Research Center,Cincinnati, Ohio) or RNAeasy (Qiagen, Valencia, Calif.). cDNA wassynthesized from 1 μg of each RNA sample using Taqman universal mastermix (Applied Biosystems, Foster City, Calif.). 10 ng was used in eachqPCR reaction. All samples were run in duplicate. Primers and probes forFoxP3 and cyclophillin A were purchased from Applied Biosystems. Realtime PCR was performed using the ABI Prism 7900 (Advanced Biosystems).FoxP3 message levels were determined after normalizing data tocyclophillin A.

Western Blotting

Nuclear extracts were prepared according to the manufacturer'sdirections using Active Motif (Carlsbad, Calif.) and 70 μg of proteinwere loaded per lane. Samples were run on NuPage 10% Bis-Tris mini-gels(Invitrogen). Proteins were transferred to PVDF membranes and incubatedwith Goat anti-FoxP3 antibody (AB2481) (Abeam, Cambridge, Mass.),followed by Rabbit anti-Goat IgG horseradish peroxidase. Blots weredeveloped with SuperSignal WestPico Chemiluminescense substrate (Pierce,Rockford, Ill.).

Cytokine Analysis

MLR culture supernatants were spun free of cells and aliquots werefrozen at −80° C. For re-stimulations, anti-CD3/CD28 beads were used ata one-to-one bead to cell ratio, or PMA at 2 ng/ml and ionomycin at 500ng/ml were used. Cells were cultured at 1 million/cells ml media in 24well plates. Supernatants were evaluated by the Luminex assay systemwith a latex bead-based multianalyte system (R&D Systems, Minneapolis,Minn.).

Monoclonal Antibodies

To evaluate purification, cells were stained with anti-CD25-PE (cloneM-A251) (BD Pharmingen, San Diego, Calif.), which is not blocked byanti-CD25-microbeads. Other antibodies for flow cytometry includedanti-CD4-PerCP (clone SK3; Becton Dickinson Immunocytometry Systems, SanJose, Calif.); anti-CD152-PE (BNI3), anti-CD27-FITC (M-T271),anti-CD62L-APC (Dreg56), anti-CD69-FITC (FN50), anti-CD134 (ACT35), from(BD Pharmingen); and anti-GITR-PE (110416), biotinylated anti-LAP(27240) from (R&D Systems). In functional experiments designed forblocking suppression, neutralizing antibodies were used at 10 μg/ml.Antibodies included anti-CTLA4 (BNI3) (BD Pharmingen), anti-PD1 (J116)(eBioscience, San Diego, Calif.), anti-OX40 (L106; Becton Dickinson),and anti-GITR (MAB689), anti-GITR-L (MAB6941), anti-OX40L (MAB10541),anti-IL10 (MAB217), anti-IL10-Receptor-_(alpha)(MAB274),anti-TGF_(beta-1,2,3) (1D11), anti-TGF_(beta-1) (MAB1835), polyclonalchicken anti-TGF_(beta/1.2) (AF-101-NA), polyclonal goatanti-TGF_(beta)RII (AF-241-NA), anti-LAP (MAB246), polyclonal goatanti-LAP (AF-246-NA), recombinant LAP (246-LP), and recombinantTGF-_(beta)RII-Ig (1003-RT; R&D Systems).

Flow Cytometry

For immunofluorescence staining, cells were stained for 30 minutes at 4°C. Cells were washed and run on a FACS Calibur cytometer (BectonDickinson). Data was analyzed by FlowJo software version 4.5 (Treestar,Ashland, Oreg.). Intracellular staining was performed using 2%paraformaldehyde fixed cells, followed by permeabilization and stainingin 0.1% saponin containing buffer.

Phenotype and FoxP3 Expression of Cord Blood Derived Suppressor Lines

Cell lines were analyzed by flow cytometry for antigens associated withsuppressor cell phenotype. Suppressor cell lines were cultured inparallel with CD4⁺CD25⁻ derived cell lines which served as conventionalT-cell controls. Cell lines were evaluated after 3 weeks of culture, atwhich time the bead based activation had nearly resolved, and the cellshad returned to a more quiescent state. The cell lines maintained arelatively stable phenotype and function for the next 3-5 weeks. Thecord derived CD25⁺ cell lines maintain remarkably uniform expression ofmultiple antigens, including CD25, intracellular CTLA4, CD27 and CD62L(FIG. 10A). In comparison, the best adult derived suppressor cell lines,generated by the most stringent purification, were slightlyheterogeneous for these antigens (FIGS. 10A and 10B).

The cord derived CD25⁺ cell lines maintained high expression of cellsurface CD25 and intracellular CTLA4, an expression pattern consideredtypical of the T_(reg) phenotype (FIG. 10A). This occurs despite thefact that other activation antigens such as CD69, CD71, and OX40 havereturned to baseline expression. The cord derived suppressor cell linesalso uniformly express both CD62L and CD27 (FIG. 10B). It was previouslydemonstrated that within potent adult derived suppressor cell lines, thecells with suppressor function reside within this double positive subset(Godfrey, et al., 2004, Blood 104: 453-461). CD25-derived cells linesalso maintain CD27 expression, but at a lower level than CD25⁺ derivedcell lines. This CD27 dim expression pattern was also noted with adultderived CD25-derived cell lines (Godfrey, et al., 2004, Blood 104:453-461).

Expression of the transcription factor FoxP3 has been proposed to be themost specific marker for regulatory cells in mice (Ramsdell, et al.,2003, Curr. Opin. Immunol., 15: 718-24). The results disclosed hereindemonstrate that the FoxP3 message was expressed at higher levels inCD25⁺ cells and lines compared to CD25⁻ T cells and lines. Freshlyisolated CD25⁺ cells from cord blood contained approximately 64 foldmore message than CD4⁺CD25⁻ cells, or fresh CD8⁺ T-cells (FIG. 11A).Cultured CD25⁺ derived cell lines contained 2-4 fold more message thanfreshly isolated CD25⁺ cells. Although message levels are low in CD25⁻cells on isolation, they increase approximately 25-30 fold on culture.This occurred, even with exhaustive depletion of CD25 dim/positive cellsprior to culture (FIG. 11A). Further re-stimulation of the 3-6 week oldcell cultures with anti-CD3/CD28 beads did not increase messageexpression, but rather slightly decreased it in both types of celllines.

In contrast to the data with mRNA message levels, western blotting witha polyclonal antibody revealed FoxP3 protein expression to be primarilyexpressed in the CD25⁺ derived cell lines. Despite expressing message,the CD25⁻ derived cell lines expressed minimal/background levels ofFoxP3 protein (FIG. 11B). Importantly, re-stimulation of the CD25⁺ celllines markedly increased FoxP3 protein expression. The increase in FoxP3protein level occurred despite the minimal change (actual decrease) inmessage levels. Re-stimulation of CD25⁻ cells still did not induce FoxP3protein in CD25⁻ derived cell lines.

The data disclosed herein demonstrates several interesting facets ofFoxP3 mRNA message and protein regulation. Most importantly, FoxP3protein was found to be relatively specific for CD25⁺ derived suppressorcells, and was minimally present in CD25⁻ derived cell lines. We alsodetermined that even exhaustively CD25 depleted CD4⁺ T-cell derivedlines could make significant amounts of FoxP3 message upon cultureactivation, approximately 20-30 fold more versus resting CD25⁻ cells.The discordance between FoxP3 mRNA and protein expression indicates thatFoxP3 message levels do not necessarily identify or quantify suppressorcells. In addition, re-stimulated CD25⁺ cells expressed much more FoxP3protein despite decreased message levels. These findings indicate FoxP3expression is augmented with activation, and suggest thatpost-transcriptional mechanisms may contribute to regulation of FoxP3protein expression.

Reactivation of Suppressor Cell Lines Induces Minimal CytokineProduction and Enhanced Surface TGF-beta LAP Expression

To determine the functional capabilities of the suppressive versusconventional T-cell lines, these cells were evaluated for theirpotential for cytokine production and cell surface molecule expressionafter re-stimulation. Cell lines were re-stimulated with anti-CD3/CD28beads for potent reactivation, and supernatants harvested at definedtime-points for analysis of cytokine content by Luminex bead basedassay. The CD25⁺ derived cell lines produced essentially no IL-2, IFN-γ,or IL-10 (FIG. 12A), while control CD25⁻ derived cell lines producedhigh levels of these cytokines. The accumulation of TNF, GM-CSF, IL-5,and the chemokine IL-8, was also markedly reduced as compared to controlcell lines (FIG. 12A). In contrast, the accumulation of TGF-beta, andthe chemokines MIP1a and RANTES were not significantly different betweendifferent cell lines.

Re-stimulation of the cell lines with pharmacological agents PMA andionomycin, which can bypass proximal signaling pathways, led to nearlyequivalent levels of cytokine production by both the CD25⁺ and CD25⁻derived cell lines (FIG. 12B). Thus, the CD25⁺ derived cell lines appearto have proximal TCR and CD28 signaling impairments that preclude thenormal production of multiple cytokines. In addition, the CD25⁺ derivedcell lines had impaired production of the immunosuppressive cytokineIL-10, which was not restored by PMA/ionomycin stimulation. Thissuggests that production of IL-10 is probably not a major mechanism ofsuppression affected by the CD25⁺ derived cell lines.

To determine if general activation of cord blood derived CD25⁺ celllines was impaired, the expression of cell surface activation antigenswas evaluated by flow cytometric analysis. Cell lines were re-stimulatedwith anti-CD3/CD28 beads and evaluated after overnight culture for CD69,OX40 (CD134), and GITR expression. All three antigens were re-expressedon both the CD25⁺ and CD25⁻ derived cell lines (FIG. 12C). Theexpression of OX40 and GITR appeared slightly enhanced on the CD25⁺derived cell lines versus the reactivated CD25⁻ cell lines (McHugh, etal., 2002, Immunity 16: 311-323). Importantly, re-activation of theCD25⁺ cell lines was relatively intact, as determined by cell surfaceactivation antigen expression analysis, in contrast to the results shownfor cytokine accumulation.

Cell surface expression of the TGF beta latency associated protein(LAP), the TGF beta precursor protein, has been reported to beassociated with T_(reg) cells (Nakamura, et al., 2004, J. Immunol., 172:834-842). In addition, recombinant forms of this protein have beenrecently reported to partially neutralize suppressor function (Nakamura,et al., 2004, J. Immunol., 172: 834-842). Thus, the expression of LAP onthe CD25⁺ and CD25⁻ derived cell lines was evaluated. Neither CD25⁺ orCD25⁻ lines expressed this protein after culture for 3-6 weeks, however,after re-stimulation with anti-C3/CD28 beads there was a distinctexpression of cell surface LAP on the CD25⁺ derived cell lines (FIG.12D), but not the CD25⁻ derived cell lines. Cell surface expression ofTGF-beta was not detectable.

Suppressor cells were found (on re-stimulation) to have limitedpotential for cytokine production. There was a profound lack of IL-2,IFN-gamma, and IL-10 production, and a markedly reduced ability toproduce GM-CSF, TNF, IL-5, and IL-8. In contrast, activation as assessedby up-regulated expression of CD69, OX40, and GITR, and production ofMIP-1a and RANTES, was generally intact. Interestingly, cytokineproduction ability was restored with PMA/ionomycin stimulation. Thisindicates a partial proximal TCR signaling block in the suppressorcells, and is consistent with the anergic response characteristics ofthe suppressor cells to TCR stimulation.

Cultured Suppressor Cells Function by an Unknown Mechanism Independentof IL-10, TGF-beta, and Multiple Costimulatory Molecules

To determine if the cultured suppressor cell lines work through knownsoluble immunosuppressive or cell surface negative regulatory proteins,DC-MLR suppressor assays were treated with neutralizing or blockingmonoclonal antibodies. Assays were evaluated for the reversal ofsuppression by resumption of proliferation. Initially antibodies toIL-10, IL-10R, and TGF-_(beta 1,2,3), were tested alone or in variouscombinations with minimal effect. Only by combining all three antibodiesat high doses (30 μg/ml), were modest and probably non-specific effectsseen (FIG. 13A). Treated control cultures also evidenced increasedproliferation. Antibodies to the negative costimulatory signalingmolecules CTLA4 and PD1 were also tested and found to have essentiallyno effect on their own, but again only in combination was a marginaleffect on suppression noted (FIG. 13B). Agonist antibodies to receptorswhose signaling is reported to abrogate or diminish suppressor functionof resting murine T_(reg), GITR (Ji, et al., 2004, J. Immunol., 172:5823-5827) and OX40 (CD134) (Takeda, et al., 2004, J. Immunol., 172:3580-3589), were tested and found to have minimal effects on thecultured suppressor cells. Agonist antibody to OX40 appeared to impairsuppressor function in some donors more than others (mean 32% reversion,n=6, range 15-75%). However, agonist antibody to OX40 also increased themagnitude of the control MLR, in approximate correlation with themagnitude of reversion of suppression (FIG. 13C). Antibodies to OX40Linhibited the MLR (with donor variability) but also increased theapparent magnitude of suppression (mean 96% suppression, n=5, range90-98%, versus 90% for control cultures) (FIG. 7C).

Because of the specific increased cell surface LAP expression onreactivated cultured suppressor cells, and the suggested importance ofTGF-beta to suppressor function (Nakamura, et al., 2004, J. Immunol.,172: 834-842; Chen, et al., 2003, Cytokine Growth Factor Rev., 14:85-89), multiple antagonists of the TGF beta pathway were evaluated.Neutralizing antibodies, both monoclonal and polyclonal, solublereceptors, recombinant LAP, and antibody to LAP were all tested in theMLR system. All reagents alone or in multiple combinations failed toreverse suppression mediated by the cultured T_(reg) cell lines.Experiments were conducted with a lower number of suppressor cells,specifically a 1:10 responder stimulator ratio, to minimize the potencyof the suppression, resulting in approximately 50% inhibition in controlMLR. Even under these conditions, minimal effects were noted (FIG. 13D).

These studies demonstrate that cord blood, when compared to adult blood,is an improved source for T_(reg) isolation and culture. Potentsuppressive cell lines were isolated from virtually every donor, andthese results were obtained with a straightforward direct MACS basedpurification. Flow cytometric profiles of antigen expression on cordblood T_(reg) cell lines were surprisingly uniform. This system was usedto further characterize suppressor cell phenotype and function.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of isolating a regulatory T cell (T_(reg) cell) from apopulation of phenotypically CD45RA⁺ blood cells, wherein said T_(reg)cell suppresses T cell proliferation, said method comprising: a)isolating a population of mononuclear cells from said human umbilicalcord blood sample; b) contacting said population of mononuclear cellswith an antibody that specifically binds CD25 under conditions suitablefor formation of a mononuclear cell-antibody complex; and c)substantially separating said mononuclear cell-antibody complex fromsaid population of mononuclear cells; thereby isolating said T_(reg)cell from a population of phenotypically CD45RA⁺ blood cells.
 2. Themethod of claim 1, wherein said population comprises an enhancedpopulation of phenotypically CD45RA⁺ blood cells, wherein said bloodcells are isolated from umbilical cord blood.
 3. The method of claim 2,wherein said umbilical cord blood sample is a human umbilical cordblood.
 4. The method of claim 2, wherein said antibody is selected fromthe group consisting of an isolated antibody, a biological samplecomprising an antibody, an antibody bound to a physical support and acell-bound antibody.
 5. The method of claim 4, wherein said antibody isselected from the group consisting of a polyclonal antibody, amonoclonal antibody, a humanized antibody, a synthetic antibody, abiologically active fragment of an antibody, and combinations thereof.6. The method of claim 5, wherein said biologically active fragment isselected from the group consisting of an Fab fragment, a F(ab′)₂fragment, a Fv fragment, and an scFv fragment.
 7. The method of claim 4,wherein said physical support is selected from the group consisting of amicrobead, a magnetic bead, an absorption column and an adsorptionmembrane.
 8. The method of claim 2, wherein said mononuclearcell-antibody complex is substantially separated from said population ofmononuclear cells by a method selected from the group consisting offluorescence activated cell sorting (FACS) and magnetic activated cellsorting (MACS).
 9. The method of claim 1, wherein steps b) and c) arerepeated.
 10. A method of multiplying a T_(reg) cell isolated by themethod of claim 2, said method comprising culturing said T_(reg) cell ina medium comprising an antibody to CD3 and an antibody to CD28.
 11. Themethod of claim 10, wherein said antibody is selected from the groupconsisting of an isolated antibody, a biological sample comprising anantibody, an antibody bound to a physical support and a cell-boundantibody.
 12. The method of claim 11, wherein said physical support isselected from the group consisting of a microbead, a magnetic bead, anabsorption column and an adsorption membrane.
 13. The method of claim10, wherein said medium further comprises IL-2.
 14. The method of claim10, wherein said T_(reg) cell expresses an antigen selected from thegroup consisting of CD25, CD4, CTLA4, CD27, CD26L and FoxP3.
 15. Amethod for inhibiting proliferation of a T cell, said method comprisingcontacting said T cell with a T_(reg) cell isolated by the method ofclaim
 1. 16. The method of claim 15, wherein said T cell is a CD4 Tcell.
 17. The method of claim 15, wherein said T cell is a CD8 T cell.18. A kit for isolating a T_(reg) cell from a human umbilical cord bloodsample, said kit comprising an antibody that specifically binds CD25bound to a physical support, an applicator, and an instructionalmaterial for the use thereof.
 19. A kit for multiplying a T_(reg) cellfrom a human umbilical cord blood sample, said kit comprising anantibody that specifically binds CD3 bound to a physical support, anantibody that specifically binds CD28 bound to a physical support, anapplicator, and an instructional material for the use thereof.
 20. AT_(reg) cell isolated by the method of claim 1.